BIOCHEMICAL CHARACTERIZATION OF PARALYTIC SHELLFISH TOXIN BIOSYNTHESIS IN VITRO

BIOCHEMICAL CHARACTERIZATION OF PARALYTICSHELLFISH TOXIN BIOSYNTHESIS IN VITRO1

Ralf Kellmann2 and Brett A. Neilan3

School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney 2052, Australia

Saxitoxin (STX) and its analogs are voltage-gatedsodium-channel blockers that cause paralytic shell-fish poisoning (PSP) and negatively affect humanhealth and seafood industries worldwide. Little isknown about the molecular biology of PSP-toxinsynthesis. Saxitoxin precursors were identified25 years ago, and a hypothetical biosynthesis path-way was proposed; however, the correct sequence ofreactions and enzymes involved in their catalysisremains to be identified. This study describes theoptimization of in vitro biosynthesis of PSP toxinsby cellular lysates of the toxic cyanobacterium Cylin-drospermopsis raciborskii (Wołosz.) Seenaya et SubbarajuT3 and the characterization of its biochemicalrequirements. Enzymes involved in PSP-toxin syn-thesis are located in the cytosol. The molecular com-ponents of in vitro biosynthesis reactions could notbe completely defined because of the requirementof an unknown cofactor. Evidence is presented thatsupports the previous suggestion that STX biosyn-thesis involves a Claisen condensation betweenarginine and acetate. In addition, carbamoyl phos-phate was identified as a likely precursor for carba-mated PSP toxins. Predictions have been maderegarding the enzymes that may be involved in thebiosynthesis of PSP toxins. These included class IIaminotransferase; nonheme iron oxygenase, contain-ing flavin, and possibly ferredoxin, as the prostheticgroups; and an O-carbamoyltransferase. On theother hand, the involvement of cytochrome P450monooxygenase was excluded.

Key index words: Cylindrospermopsis raciborskii;in vitro biosynthesis; inhibitor; paralytic shellfishtoxins; precursor

Abbreviations: CARBP, carbamoylphosphate; LC–MS, liquid chromatography–mass spectroscopy; PSP,paralytic shellfish poisoning; STX, saxitoxin

Paralytic shellfish poisoning (PSP) is a life-threatening affliction that results from the con-sumption of drinking water or seafood that iscontaminated by saxitoxin (STX) and its analogs.

These toxins interfere with the function of at leastthree types of voltage-gated ion channels, such asneuronal sodium channels (Kao and Levinson1986), as well as calcium and potassium channelsthat are important for the functioning of heartmuscle (Wang et al. 2003, Su et al. 2004). Paralyticshellfish poisoning toxins are produced by bothfreshwater and marine organisms that span twokingdoms of life. Several species of marine dino-flagellates belonging to the genera Alexandrium,Pyrodinium, and Gymnodinium are capable of produ-cing PSP toxins (Cembella 1998). They are thecause of an estimated 2000 annual cases of PSP inhumans globally (Hallegraeff 1995). In freshwaterenvironments, several filamentous species of cyano-bacteria from the genera Anabaena, Aphanizomenon,Cylindrospermopsis, and Lyngbya are known to pro-duce PSP toxins and are considered a serious toxi-cological health risk that may affect humans,animals, and ecosystems worldwide (Sivonen andJones 1999, Chorus et al. 2000, Carmichael 2001,Haider et al. 2003).

Saxitoxin and its analogs are perhydropurinealkaloids that share a tricyclic carbon backbone butdiffer in their functional groups at four positions(Table 1). Although STX may structurally resemblethe purines of primary metabolism, a series ofdetailed labeled-precursor feeding studies hasrevealed a complex and unique biosynthetic path-way (Fig. 1; Shimizu 1993). The first product of bio-synthesis is believed to be STX, which may bemodified at four different positions to producemore than 20 STX analogs (Table 1). Although theprecursors and some of the biochemical mecha-nisms involved in STX biosynthesis have been inves-tigated, the sequence of reactions and the identityof intermediates have remained hypothetical andawait identification.

Attempts that have been made so far to identifythe genes and enzymes that are involved in thebiosynthesis of PSP toxins have been largely unsuc-cessful (Taroncher-Oldenburg and Anderson 2000,Sako et al. 2001, Yoshida et al. 2002, Pomati et al.2004a, Pomati and Neilan 2004). The main difficul-ties to overcome include the low, naturally occur-ring concentrations of the toxins; the lack ofsensitive, high-throughput methods for their detec-tion; and the lack of methods for the genetic mani-pulation of PSP-toxin-producing microorganisms.

1Received 19 April 2006. Accepted 23 February 2007.2Present address: Department of Molecular Biology, The Univer-

sity of Bergen, Bergen 5008, Norway.3Author for correspondence: e-mail [email protected].

J. Phycol. 43, 497–508 (2007)� 2007 Phycological Society of AmericaDOI: 10.1111/j.1529-8817.2007.00351.x

497

The goal of this study was to increase knowledgeregarding PSP-toxin biosynthesis that may eventuallylead to the identification of STX biosynthesisenzymes and genes. The biochemical requirementsfor PSP-toxin production were determined using anin vitro PSP-toxin biosynthesis assay. Finally, we des-cribe a cellular cofactor that was only present in thePSP-toxin-producing strain Cylindrospermopsis raciborskiiT3 and that stimulated the production of PSP toxinsin vitro. A novel hypothesis regarding the nature ofSTX biosynthesis enzymes is presented.

MATERIALS AND METHODS

Organisms and culturing conditions. The filamentous freshwa-ter cyanobacterium C. raciborskii T3 was kindly provided bySandra Azevedo (Federal University of Rio de Janeiro, Brazil).This strain was isolated in Brazil from a toxic cyanobacterialbloom in the State of Sao Paulo (Lagos et al. 1999). The strainC. raciborskii AWT205 was obtained from Peter R. Hawkins(Sydney Water Corporation, West Ryde, Australia). All cyano-bacteria were grown in Jaworski medium (Thompson et al.

1988) in static batch culture at a constant temperature of 26�Cand with continuous irradiance by cool-white light from afluorescent tube at an intensity of 10 lmol photon Æ m)2 Æ s)1.Escherichia coli DH5a was grown overnight in Luria broth at37�C with shaking (120 rpm).

Extraction of proteins. All procedures were carried out at 4�Cunless otherwise stated. One liter of late-exponential-phaseC. raciborskii culture (OD750 = 0.3) was harvested by gentlecentrifugation (1000g for 20 min). The cell pellet was washedtwice with sterile 10 mM Tris pH 8.5 and resuspended in 5 mLof the same buffer. The cell suspension was passed once ortwice through a French press (Paton Scientific Pty. Ltd., WaggaWagga, Vic., Australia), which achieved near-complete cell lysis.The degree of cell lysis was verified by microscopy. Cell debriswas removed from the lysate by centrifugation at 12,000g for10 min. To remove any remaining particulates, the supernatantwas filter-sterilized through a disposable syringe filter(0.22 lm). The protein extract obtained was aliquoted, snap-frozen in liquid nitrogen, and stored at )80�C until use. Itcontained approximately 1.5 mg Æ mL)1 total protein.

Desalted protein extracts were prepared by gel filtrationusing Sephadex G-10 (Amersham Bioscience Cat-No.: 17-0010-01, Castle Hill, NSW, Australia). Protein extracts were desaltedagainst a buffer containing 50 mM Tris pH 8.5 and 25 mMNaCl. Desalted enzyme extracts were assayed for in vitro PSP-toxin biosynthesis activity using optimized reaction conditions(as described below).

Cytosolic and membrane fractions of C. raciborskii T3 lysateswere prepared by centrifugation at 100,000g for 1 h. Theefficiency of separation was judged visually by the removal ofchl, which is membrane bound, from the supernatant. In caseswhere green pigment was still visible, centrifugation wasrepeated for another 1 h. The supernatant was divided intothree fractions from top to bottom (Fig. 12). The membranepellet was washed once and resuspended in a small volume ofbuffer (50 mM Tris pH 8.5). Resuspension of the pellet wasaided by sonication for 30 s using a Branson ultrasonic celldisruptor (Consonic Pty. Ltd., Seven Hills, NSW, Australia) thatwas equipped with a microtip and set to an amplitude of 25%with a duty cycle of 50%. Each fraction was assayed for in vitroPSP-toxin biosynthesis activity using optimized reaction condi-tions, described below.

Extraction of unknown cofactor required for PSP-toxin biosynthesis. Theresuspended membrane pellet was extracted with solvents(choloroform, acetone, and methanol) of increasing polarity toinvestigate the solubility of the unknown cofactor. The solventswere added to the membrane fraction at a ratio of 1:1. Aftervigorous mixing, the extracts were centrifuged at 12,000g for15 min. Supernatants and pellets were collected, and wheresolvents and water were immiscible, the aqueous and organicphases were separated. After evaporation of the solvent byvacuum centrifugation, the pellets were redissolved in 50 mMTris buffer (pH 8.5). The solutions were then used in theoptimized in vitro PSP-toxin biosynthesis reaction containingdesalted enzyme extract.

In vitro biosynthesis of PSP toxins. To determine the optimumpH for in vitro biosynthesis of PSP toxins, reactions were adjustedto final pH values of 5, 6, 6.9, 8, 9, and 10 with 100 mM of thefollowing buffers: acetate pH 5; 2-morpholinoethanesulfonicacid pH 6; Tris(hydroxymethyl)aminomethane (Tris) pH 6.9and 8; 2,2-(cycloamino)ethanesulfonic acid (CHES) pH 9; and3-(cyclohexylamino)-1-propanesulfonic acid pH 10. In vitroPSP-toxin biosynthesis was carried out in 18 lL reactionvolumes containing final concentrations of 50 mM Tris pH8.5; 200 mM NaCl; 4% Tween-80; 1 mM ATP; 1 mM MgCl2;0.05 mM NADH; 0.05 mM NADPH; and 1 mM each of argin-ine, S-adenosylmethionine (SAM), acetyl-CoA, and carbamoyl-phosphate (CARBP) as substrates. For greater convenience, athree times concentrated reaction buffer stock solution was

Table 1. Molecular structure of paralytic shellfish poison-ing toxins (modified from Schantz et al. 1975).

N

NNH

NH

+H2N

NH2+

R4

R3

OH

R1

R2

OH

1

23

4

56

10 11

12

78

9

13

R-1 R-2 R-3 R-4 Compound name

Carbamates –H –H –H STX–OH –H –H neoSTX–OH –OSO3

) –H GTX-1–H –OSO3

) –H GTX-2–H –H –OSO3

) GTX-3–OH –H –OSO3

) GTX-4N-sulfocarbamates –H –H –H GTX-5

–OH –H –H GTX-6–OH –OSO3

) –H C-3–H –OSO3

) –H C-1–H –H –OSO3

) C-2–OH –H –OSO3

) C-4Decarbamoyl toxins

–OH

–H –H –H dcSTX–OH –H –H dcneoSTX–OH –OSO3

) –H dcGTX-1–H –OSO3

) –H dcGTX-2–H –H –OSO3

) dcGTX-3–OH –H –OSO3

) dcGTX-4Deoxy toxins

–H

–H –H –H doSTX–OH –OSO3

) –H doGTX-2–OH –H –OSO3

) doGTX-3

C, C-toxin; dc, decarbamoyl; do, deoxy; GTX, gonyautoxin;STX, saxitoxin.

498 RALF KELLMANN AND BRETT A. NEILAN

prepared and stored at )80�C until use. All reactions wereprepared on ice. A reaction was prepared by mixing 6 lL of 3·reaction buffer with 4.5 lL crude enzyme extract. Dependingon the experiment, a measured volume of test compound was

added. The volume of the reaction was finally adjusted to 18 lLwith Milli-Q-purified water (Millipore Australia Pty. Ltd., NorthRyde, NSW, Australia). In vitro PSP-toxin biosynthesis reactionswere incubated for 1 h in a water bath at 35�C under cool-white

Fig. 1. Biosynthetic pathway for saxitoxin (modified from Shimizu 1993). (1) Claisen condensation between arginine and acetate; (2)amidinotransfer from a second arginine to a-amino group of first intermediate; (3) formation of the first heterocycle by a retro-aldol-likecondensation; (4) formation of the remaining two heterocycles by unknown reactions; (5) introduction of methyl side chain by electrophi-lic attachment; (6) rearrangement involving 1,2-H shift from C-6 to C-5; (7) epoxidation of methyl side chain; (8) opening of epoxide toan aldehyde; (9) reduction of aldehyde to alcohol; (10) O-carbamoyl transfer and oxidation reactions completing the STX molecule. =Xmay be two hydrogens, a keto, or two hydroxy groups.

IDENTIFICATION OF CANDIDATE SAXITOXIN BIOSYNTHESIS ENZYMES 499

light (10 lmol Æ m)2 Æ s)1) from a fluorescent tube, unlessotherwise stated. Reactions were stopped by placing them onice and adding 2 lL of 5 M acetic acid. The reactions wereimmediately processed for the measurement of PSP toxins byHPLC.

High-pressure liquid chromatography and liquid chromatography–mass spectroscopy. Calibration standards for PSP toxins (PSP-1Bkit) were obtained from the Canadian National ResearchCouncil. Screening for PSP toxins was performed by prechro-matographic oxidation with hydrogen peroxide followed byHPLC separation. Chromatography was carried out accordingto the method by Lawrence et al. (1996). Briefly, chemicalanalyses were performed using a Hewlett & Packard Series 1100HPLC apparatus coupled to a Hewlett and Packard 1040fluorescence detector (Agilent Technologies, Forest Hill, Vic.,Australia). The column was a Supelcosil LC-18(150 mm length · 4.6 mm inner diameter, 5 lm particle size;Phenomenex, Lane Cove, NSW, Australia). The samples andstandard mixture were oxidized as previously described (Law-rence et al. 1996) using hydrogen peroxide prior to injection.Oxidation products (25 lL injection volume) were elutedunder isocratic conditions with 2% acetonitrile (v ⁄ v) in 0.1 Mammonium formate, pH 6.0, at a flow rate of 1.1 mL Æ min)1.To verify the oxidation dependency of HPLC peaks, hydrogenperoxide was removed from the oxidant solution. The concen-tration of individual STX analogs could not be determined byHPLC with prechromatographic oxidation, as it may yield thesame oxidation products from different toxin analogs (Law-rence et al. 1996). Because the fluorescent yields of theoxidation products from PSP toxins were approximately equal,the total concentration of PSP toxins was calculated based onthe combined peak areas and the fluorescent yield of theoxidation product from STX. The production of toxins wasexpressed as the change in the total concentration of PSPtoxins over 1 h.

Cyanobacterial extracts and PSP-toxin standards were ana-lyzed using a Thermo Finnigan Surveyor HPLC and autosam-pler that was coupled to a Thermo Finnigan LCQ Deca XP Plusion trap mass spectrometer (Thermo Fisher Scientific, SanJose, CA, USA) fitted with an electrospray source. Separation ofanalytes was obtained on a 2.1 mm · 150 mm PhenomenexLuna 3 micron C18 column (Phenomenex Inc., Torrance, CA,USA) at 100 lL Æ min)1. Analysis was performed using agradient starting at 95% aqueous, 10 mM heptafluorobutyricacid in water. This was held for 10 min and then ramped to100% organic (acetonitrile) over 30 min. The gradient washeld at 100% B for 10 min to wash the column and thenreturned to 100% A and again held for 10 min to equilibratethe column for the next sample. This resulted in a run time of60 min per sample. Sample volumes of 10–100 lL wereinjected for each analysis. The HPLC eluate directly enteredthe electrospray source, which was set up as follows: electro-spray voltage, 5 kV; sheath gas-flow rate, 30 arbitrary units;auxiliary gas-flow rate, five arbitrary units. The capillarytemperature was 200�C and had a voltage of 47 V. Ion opticswere optimized for maximum sensitivity before sample analysisusing the instrument’s autotune function with a standard toxinsolution. Mass spectra were acquired in the centroid mode overthe m ⁄ z range 145–650. Mass range setting was ‘‘normal,’’ with200 ms maximum ion injection time and automatic gaincontrol on. Collision energy was typically 20–30 ThermoFinnigan arbitrary units and was optimized for maximalinformation using standards.

RESULTS

In vitro biosynthesis of PSP toxins. The productionof PSP toxins by strains of C. raciborskii has been

previously reported (Lagos et al. 1999, Molica et al.2002). Analysis of cellular extracts fromC. raciborskii T3 by liquid chromatography–massspectroscopy revealed the presence of STX, neosaxi-toxin (neoSTX), and decarbamoylsaxitoxin (dcSTX;Fig. 2). Analysis by HPLC yielded five oxidation-dependent peaks with retention times of 3.5, 3.8,6.4, 6.9, and 16.2 min. These peaks were indistin-guishable from those produced by calibration stand-ards for STX, dcSTX, and neoSTX (Fig. 3). TheHPLC analysis of C. raciborskii AWT205, which doesnot produce any PSP toxins, did not yield any peakswith retention times that were similar to thoseof the calibration standards or extract fromC. raciborskii T3. The mean coefficient of variationof 28 triplicate HPLC measurements was 0.041(±0.026 SD, minimum 0.004 and maximum 0.096),which demonstrated the high reproducibility ofHPLC measurements.

Initially, a 0.22 lm filtered raw lysate ofC. raciborskii T3 and the same lysate that had beenboiled for 10 min, which thus served as a control,were incubated for 24 h at 26�C under standardculturing conditions. Prior to incubation, the lysatecontained 1577 nM (±195 SD) of PSP toxins, whichincreased by 17% to 1843 nM (±161 SD) over thefirst 3 h, and by 55% to 2447 nM (± 207 SD) after24 h of incubation. The concentration of PSPtoxins in the boiled control reactions decreased by10 nM and did not represent a significant change.When cell lysate was supplemented with 1 mMarginine or 1 mM CARBP, in vitro PSP-toxin pro-duction was, respectively, 5.2% and 11.2% higherthan in nonsupplemented controls. Supplementa-tion with 1 mM acetyl-CoA or 1 mM SAM had littleeffect (respectively, )4.3% and )2.1% change inthe amount of PSP toxins produced; Fig. 4). Pre-cursor concentrations of more than 1 mMdecreased PSP-toxin production in comparison withuntreated lysate, with the exception of SAM, whichstimulated PSP-toxin production up to concentra-tions of 100 mM (Fig. 4). Addition of 1 mM of allfour precursors in combination increased PSP-toxinproduction by 40.8% over the control, which wasgreater than the sum of all precursors usedindividually at a concentration of 1 mM (Fig. 4).In vitro PSP-toxin biosynthesis increased linearlywith increasing ionic strength up to a concentra-tion of 200 mM for NaCl and 150 mM for KCl, aswell as with increasing concentration, up to 4%, ofthe detergent Tween-80.

A large increase in the in vitro production of PSPtoxins by up to 65% was observed when reactionswere supplemented with the artificial electron car-rier methylviologen (Fig. 5; Table 2), whereasNADH and NADPH moderately stimulated the pro-duction of PSP toxins (11% and 17%, respectively;Table 2). The ATP (1 mM) in the presence of equi-molar concentrations of MgCl2 had little effect


Fig. 2. Liquid chromatography–mass spectroscopy (LC–MS) ana-lysis of cellular extracts fromCylindrospermopsis raciborskii T3.The upper traces representselective ion monitoring at theindicated m ⁄ z ratios. Below eachtrace, an MS ⁄ MS fragmentationspectrum at the indicated retent-ion time is given. Values of m ⁄ zratios are given for diagnosticfragment ions according to Slenoet al. (2004) and Fang et al.(2004). (a) Analysis for saxitoxin(STX; m ⁄ z 300); (b) Analysis forneosaxitoxin (neoSTX; m ⁄ z 316);(c) Analysis for decarbamoylsaxi-toxin (dcSTX; m ⁄ z 257).


(5.5% increase over the control; Table 2). In vitroPSP-toxin production occurred over broad ranges ofpH and temperature. High variability was observedin the production of PSP toxins over the pH rangefrom 5 to 10 (Fig. 6), where it had a plateau betweenpH 7 and 9. In vitro PSP-toxin production occurredover a temperature range from 23 to 38�C and wasthe highest at 35�C (Fig. 7). Interestingly, the pro-duction of PSP toxins was greatly reduced in thedark, where the concentration increased linearly atlow levels over 6 h (Fig. 8). In contrast, the biosyn-thesis rate was 12 times greater in light over the firsthour and decreased to only approximately 1.6 timesthe rate of the dark reaction for the remaining 5 h.

In vitro PSP-toxin biosynthesis under optimized condi-tions. Based on the results described above, an opti-mized in vitro PSP-toxin biosynthesis reaction was

designed. The optimized reaction buffer contained50 mM Tris pH 8.5; 200 mM NaCl; 4% Tween-80;1 mM ATP; 1 mM MgCl2; 0.05 mM each of NADHand NADPH; 1 mM methylviologen; and 1 mMeach of arginine, acetyl-CoA, SAM, and CARBP.Optimized reaction conditions were 35�C withirradiation by cool-white light at an intensity of10 lmol photons Æ m)2 Æ s)1. In vitro PSP-toxin bio-synthesis under standard conditions was measuredover 2 h at 20 min intervals (Fig. 9). The concentra-tion of PSP toxins in the reaction increased by 69%over 2 h. All three toxin peaks detected by HPLCincreased proportionally.

–30

–20

–10

0

10

20

30

40

50

arginine acetyl-CoA SAM CARBP all combined

PS

P-t

oxin

pro

duct

ion

rela

tive

to c

ontr

ol w

ithou

t ad

ded

prec

urso

rs (

%)

1 mM 10 mM 100 mM

Fig. 4. Effect of precursor concentration on the in vitro pro-duction of paralytic shellfish poisoning (PSP) toxins in terms ofpercentage relative to a raw lysate without addition of precursors.Error bars indicate the standard deviation of measurements.CARBP, carbamoylphosphate; SAM, S-adenosylmethionine.

Fig. 5. Effect of methylviologen on the production of PSP tox-ins in Cylindrospermopsis raciborskii T3 cellular lysate. Error barsindicate the standard deviation of measurements.

Fig. 3. HPLC chromatograms of Cylindrospermopsis raciborskiiT3 and calibration standards. dcSTX, decarbamoylsaxitoxin;neoSTX, neosaxitoxin; and STX, saxitoxin.

Table 2. Effect of various cofactors, substrates, and inhib-itors on paralytic shellfish poisoning toxin biosynthesis.

Compound Concentration (mM) Inhibition (%) Activation (%)

ATP ⁄ MgCl2 1 – 6NADPH 0.05 ) 17NADH 0.05 ) 11Methylviologen 1 ) 65Agmatine 1 29 )Canavanine 1 17 )Guanidine 1 39 )Spermine 1 21 )Ornithine 1 39 )Hydroxylamine 0.1 59 )Hydroxylamine 1 93 )CO 10 min purging ) 51a-ketoglutarate 0.5 7 )

1 63 )10 94 )

Mg2+ 1 ) 11Ca2+ 1 2 )EDTA 10 30 )Fe3+ 1 68 )Fe2+ 1 52 )Ni2+ 1 47 )Cu2+ 1 69 )Co2+ 1 24 )Mn2+ 1 39 )


Inhibitors and activation of PSP-toxin biosynthesis. Arange of compounds was screened for their effectson in vitro PSP-toxin biosynthesis under optimizedconditions (Table 2). Analogs of arginine (agma-tine, canavanine, guanidine, spermine, and ornith-ine) inhibited PSP-toxin biosynthesis, as expected ifarginine was the correct substrate. Hydroxylamine isan inhibitor of pyridoxal phosphate (PLP)–depend-ent enzymes and strongly inhibited PSP-toxin bio-synthesis (59% at 0.1 mM). Carbon monoxide is astrong but reversible inhibitor of cytochrome P450–dependent enzymes, which have been proposed tobe involved in PSP-toxin biosynthesis. The strong sti-mulation of PSP-toxin production by carbon monox-ide (51% increase over the control) was thereforeunexpected (Fig. 10). To exclude the possibility thatlight reversed any inhibition by carbon monoxide,the experiment was repeated in the dark and in the

Fig. 7. Temperature dependency of in vitro production ofparalytic shellfish poisoning (PSP) toxin in Cylindrospermopsis raci-borskii T3 cellular lysate. The data points represent the concentra-tion of PSP toxins after incubation for 1 h. The data point at 0�Crepresents incubation on ice. Error bars indicate the standarddeviation of measurements.

Fig. 6. pH dependency of in vitro production of paralyticshellfish poisoning (PSP) toxin in Cylindrospermopsis raciborskii T3cellular lysate. The data points represent the concentration ofPSP toxins after incubation for 1 h. Error bars indicate the stand-ard deviation of measurements.

0

500

1000

1500

2000

2500

3000

0 1 2 3 4 5 6Time (h)

PS

P-t

oxin

con

cent

ratio

n (n

M)

Light Dark

Fig. 8. Light-dependent regulation of in vitro biosynthesis ofparalytic shellfish poisoning (PSP) toxins in Cylindrospermopsisraciborskii T3 cellular lysate. The data points represent theconcentration of PSP toxins after incubation for 1 h. Error barsindicate the standard deviation of measurements.

Fig. 9. In vitro biosynthesis of paralytic shellfish poisoning(PSP) toxins under standardized conditions in Cylindrospermopsisraciborskii T3 cellular lysate. The reaction buffer contained 50 mMTris pH 8.5; 4% Tween-80; 200 mM NaCl; 1 mM each of arginine,acetyl-CoA, S-adenosylmethionine (SAM), carbamoylphosphate(CARBP), methylviologen, ATP, and MgCl2; 0.05 mM each ofNADH and NADPH. Error bars indicate the standard deviation ofmeasurements.

Fig. 10. Effect of carbon monoxide and air on in vitro produc-tion of paralytic shellfish poisoning (PSP) toxins in Cylindrospermop-sis raciborskii T3 cellular lysate in light and in darkness for 120 min.Error bars indicate the standard deviation of measurements.


light. Control reactions were purged with air for thesame duration and at the same flow rate as carbon-monoxide-treated reactions to compensate for anypossible evaporation during purging. PSP-toxin bio-synthesis rates in the light were 4.8 times and 3.5times the rate in the dark after purging with air andcarbon monoxide, respectively (Fig. 12). Carbonmonoxide strongly stimulated in vitro PSP-toxin pro-duction both in light (43% increased productionover the control) and in darkness (58% increasedproduction over the control). 2-Oxoglutarate (2OG)is the substrate for 2OG–dependent dioxygenases;however, it inhibited in vitro PSP-toxin biosynthesis.Magnesium is required by enzymes, such as ATPhydrolyzing enzymes; however, calcium may replacemagnesium in certain enzymes. PSP-toxin biosynthe-sis was stimulated in the presence of 1 mM magnes-ium, but calcium had no effect at the sameconcentration. The EDTA inhibited the in vitroPSP-toxin biosynthesis moderately at a concentra-tion of 10 mM. Other metal ions tested (Table 2)form part of the redox centers in certain enzymes;however, they inhibited PSP-toxin biosynthesis.

Cellular location of PSP-toxin biosynthesis enzymes. High-speed centrifugation of C. raciborskii T3 lysate(100,000g for 1 h) efficiently separated membranesfrom the cytosolic fraction. Water-soluble phycocya-nin gave the supernatant a deep blue color, whilewater-insoluble chl gave the membrane pellet a darkgreen color. PSP-toxin biosynthesis and the startingconcentration of PSP toxins in the fractionatedlysate were not homogenously distributed aftercentrifugation (Fig. 11). All three soluble fractions

(supernatants I to III) showed activity. The activitydetected in supernatant I was 21% of that in super-natant II, which provided the highest activity, fol-lowed by supernatant III, which provided 84% theactivity of supernatant II. Supernatant III was the cyto-solic fraction with the highest starting concentrationof PSP toxins, while the initial concentration of PSPtoxins in supernatants I and II was 77% that of super-natant III. Approximately 50% of the total PSP-toxincontent in the cell lysate sedimented with the mem-brane fraction; however, no PSP-toxin biosynthesisactivity was detected in the membrane fraction.

Minimal biochemical requirements of PSP-toxin produc-tion. Desalting, using a Sephadex G-10 column(Amersham Bioscience Cat-No.: 17-0010-01, CastleHill, NSW, Australia), removed approximately 93%of PSP toxins from the C. raciborskii T3 proteinextract and abolished in vitro PSP-toxin biosynthesisactivity (Fig. 12). A range of common cofactors(NAD, NADP, FAD, FMN, PLP, thiamine pyrophos-phate, and biotin) and metal ions (K, Na, Rb, Mg,Ca, Fe, Ni, Co, and Mo) were tested individuallyand in all possible combinations in an attempt toidentify the missing cofactors required for in vitroPSP-toxin biosynthesis. Biosynthetic activity, how-ever, could not be restored to the desalted extract.To determine whether the unknown compoundessential to STX biosynthesis was unique toC. raciborskii T3 or present in other microorganisms,in vitro reactions with desalted C. raciborskii T3 pro-tein extract were supplemented with the salt frac-tion and untreated lysate from the non-PSP-toxin-producing microorganisms C. raciborskii AWT205,

Fig. 11. Subcellular fractionation of Cylindrospermopsis racibor-skii T3 lysate. S ⁄ N, supernatant.

–200

0

200

400

600

800

1000

1200

1400

1600

1800

2000

T3dT3m

T3d+T

3s

T3d+T

3m

T3d+T

3mCHL-

AQ

T3d+T

3mACT

T3d+T

3mM

ET

T3d+A

WTs

T3d+P

CC7120

s

T3d+E

colis

PS

P-t

oxin

pro

du

ctio

n (

nM

·h–1

)

Fig. 12. Restoration of in vitro biosynthesis activity of para-lytic shellfish poisoning (PSP) toxin of desalted Cylindrospermopsisraciborskii T3 cellular lysates by supplementation with variouscellular extracts. T3d, desalted protein extract from C. raciborskiiT3; T3s, salt fraction from C. raciborskii T3 cellular extract; T3m,membrane fraction from C. raciborskii T3; T3mCHL-AQ, aqueousfraction after chloroform extraction of C. raciborskii T3 membranefraction; T3mACT, acetone extract of C. raciborskii T3 membranefraction; T3mMET, methanol extract of C. raciborskii T3 mem-brane fraction; AWTs, salt fraction of C. raciborskii AWT205 cellu-lar extract; PCC7120s, salt fraction of Anabaena PCC7120 cellularextract; Ecolis, salt fraction of Escherichia coli DH5a cellularextract.


Anabaena sp. PCC7120, and E. coli DH5a. None ofthese extracts could restore PSP-toxin biosynthesisactivity. Activity was restored to approximately 66%of untreated C. raciborskii T3 protein extract afterthe addition of an equivalent volume of the saltfraction of C. raciborskii T3 to the protein extract.Activity in reactions with desalted C. raciborskii T3protein extracts was also restored by supplementa-tion with the C. raciborskii T3 membrane fraction.The stimulatory effect of the membrane fraction onin vitro PSP-toxin biosynthesis activity was also main-tained after boiling for 15 min. No activity wasobserved in reactions containing only the mem-brane fraction (Fig. 12). The unknown cofactor thatwas essential for in vitro PSP-toxin biosynthesiscould be extracted from the membrane fractionwith polar solvents. The greatest capacity to restorePSP-toxin biosynthesis activity of the desaltedextract was found in the aqueous phase after extrac-tion of the membrane fraction with chloroform, fol-lowed by methanol and acetone extracts indecreasing order (Fig. 12). In addition to theunknown cofactor, PSP toxins were coextracted tovarying degrees by these solvents; however, the stim-ulatory activity measured for the extracts was notrelated to the amount of coextracted PSP toxins(data not shown).

DISCUSSION

Precursors for PSP-toxin biosynthesis were identi-fied more than two decades ago, and a hypotheticalbiosynthesis pathway was proposed (Shimizu 1993).Based on this pathway, speculation regarding theidentity of enzymes involved in STX biosynthesis hasbeen presented. Since then, the activities of twosulfotransferases from the dinoflagellate Gymnodiniumcatenatum L. W. Graham have been described, whichare presumably involved in the biotransformation ofSTX into GTX-2 ⁄ 3, GTX-5, and C-1 ⁄ 2 toxins (Sakoet al. 2001, Yoshida et al. 2002). In those studies, nosequence information could be obtained because ofthe low yield of active enzymes during purification.Other studies have attempted to identify STX bio-synthesis genes using comparative transcriptomicand genomic methods, including differential display(Taroncher-Oldenburg and Anderson 2000), PCR-based positive subtractive hybridization (Pomati andNeilan 2004), and comparative HIP-PCR (highly iter-ated octameric palindrome repeated-sequence PCR;Pomati et al. 2004a); however, the identification of agene directly involved in the biosynthesis of STXremains elusive. The natural concentrations of PSPtoxins are in the nanomolar range, and correspond-ingly, the levels of STX biosynthesis gene transcriptsand enzymes may also be very low. Producing micro-organisms are difficult to culture; they grow slowlyand do not reach high cell densities. Most import-antly, these organisms are not amenable to geneticmanipulation. In view of these difficulties and the

lack of progress to identify any of the STX biosyn-thesis genes, there is a need for more detailedknowledge of STX biosynthesis.

In vitro PSP-toxin biosynthesis. The advantage ofin vitro biosynthesis is that selectively permeablemembranes do not need to be crossed by added com-pounds, and therefore the chemical composition ofthe reaction can be defined. The in vitro biosynthe-sis of PSP toxins by lysates of C. raciborskii T3 hasbeen reported (Pomati et al. 2004b) and was there-fore regarded as a suitable tool to determine thebiochemical requirements and inhibitors of PSP-toxin biosynthesis. In the study by Pomati et al.(2004b), no efforts were made to determine opti-mum conditions for in vitro PSP-toxin biosynthesis.In the present study, the concentration of PSP tox-ins in untreated C. raciborskii T3 lysate increased by17% (266 nmol Æ L)1) after 3 h and by 55%(870 nmol Æ L)1) after 24 h of incubation understandard culturing conditions. Under optimizedconditions, the concentration of PSP toxins in thereaction mix increased by 69% after 2 h incubation.All three toxin peaks increased at proportionalrates, indicating that the observed production wasnot because of chemical interconversion of toxinanalogs. Contaminating bacteria or cyanobacterialcells as possible sources for the observed toxinincrease were also excluded, as the protein extractwas filter-sterilized. On the other hand, in vitro PSP-toxin production provided the characteristics thatare typical of enzymatic reactions. All activity ceasedafter boiling the extract for 5 min, and PSP-toxinproduction occurred within pH and temperaturelimits that are typical for enzymes.

Inhibitors and activators of PSP-toxin biosynthesis. Theidentity of acetyl-CoA and SAM as direct precursorsof PSP toxins has been demonstrated; however,uncertainty remained as to whether arginine, agma-tine, or ornithine was the direct precursor in thefirst step of synthesis (Fig. 1) and what the origin ofthe O-linked carbamoyl side chain was (Shimizu1993). Identification of the direct precursors willincrease the chances of correctly predicting theidentity of PSP-toxin biosynthesis enzymes. Shimizu(1993) proposed that the first step in PSP-toxin bio-synthesis is a Claisen condensation between acetateand arginine or ornithine (step 1, Fig. 1). Claisencondensations on amino acids are rare in nature,and only one enzyme family is known to performsuch reactions, which are the class II aminotransf-erases (Eliot and Kirsch 2004). Alternative to theproposed Claisen condensation, PSP toxins may beproduced in an analogous fashion to nicotine bio-synthesis, where arginine is decarboxylated by theaction of arginine decarboxylase to produce agma-tine, which is subsequently utilized to produce nico-tine (Ghosh 2000). In PSP-toxin biosynthesis,arginine may be decarboxylated, and the resultingagmatine condensed to acetate by an acetyl transf-erase. O-carbamoyl groups are present in a wide


range of bacterial secondary metabolites, suchas antibiotics (Coque et al. 1995) and rhizobialnodulation factors (Jabbouri et al. 1998). Theyare invariably formed by a member of theO-carbamoyltransferase enzyme family, which trans-fers carbamate from CARBP to a free hydroxylgroup. Carbamoylphosphate may therefore be an asyet unidentified precursor in PSP-toxin biosynthesis.

In vitro PSP-toxin biosynthesis was strongly stimu-lated (40.8%) only when reactions were supplemen-ted with arginine, acetyl-CoA, SAM, and CARBP incombination (Fig. 3). These results indicated thatall four compounds are required for PSP-toxin bio-synthesis. The identity of arginine as the correctprecursor was further supported by the inhibition ofin vitro PSP-toxin biosynthesis by agmatine andornithine (29% and 39% inhibition, respectively) instandard reactions, which contained all four precur-sors. These data are in support of the proposal thata class II aminotransferase, catalyzing a Claisen con-densation between arginine and acetate, is involvedin PSP-toxin biosynthesis. In addition, the identifica-tion of CARBP as a likely precursor suggests that anO-carbamoyltransferase is also involved.

Class II aminotransferases are PLP-dependentenzymes and are specifically inhibited by hydroxyl-amine (Mukherjee and Dekker 1987). The stronginhibition (59%) of in vitro PSP-toxin biosynthesisby 0.1 mM hydroxylamine supported the sugges-tions that a PLP-dependent enzyme is involved; how-ever, PSP-toxin biosynthesis may involve twodifferent PLP-dependent enzymes. A reaction likelyto follow amidination of PSP-toxin precursor (step2, Fig. 1) is the formation of the first ring by aninternal condensation between the amine from thenewly introduced amidino group and the acetate-derived carbonyl carbon (step 3, Fig. 1). Thisreaction is similar to the cyclization of b-hydroxyarginine in capreomycidine biosynthesis (Thomas etal. 2003), which is catalyzed by the PLP-dependentaminotransferase, VioD. Hydroxylamine may thushave inhibited step 1 and ⁄ or step 3 of PSP-toxinbiosynthesis (Fig. 1).

Most of the energy required for PSP-toxin biosyn-thesis may be derived from activated compounds,such as acetyl-CoA and SAM. Other biosyntheticsteps, however, may require energy in the form ofATP or may be regulated by ATP. Addition of ATPto in vitro reactions had little effect (6% stimula-tion) on PSP-toxin biosynthesis (Table 2), indicatingthat it was either present at nonlimiting concentra-tions in in vitro biosynthesis reactions or notrequired for STX biosynthesis.

The biosynthesis of PSP toxin involves several oxi-dative steps. Two of the three heterocycles areformed by an unidentified oxidative mechanism(step 4, Fig. 1), and several positions of PSP toxinsmay be hydroxylated (Table 1). In bacteria, at leastthree types of oxygenases are involved in the oxida-tion of secondary metabolites, which are the

cytochrome P450–dependent monooxygenases,flavin-dependent dioxygenases, and 2OG dioxygen-ases. The reaction mechanism involved in thehydroxylation of the methyl side chain (steps 7–9,Fig. 1) is typical of cytochrome P450–dependentmonooxygenases (Bernhardt 2006). Carbon monox-ide is a specific inhibitor of cytochrome P450, andits inhibitory effect is reversible by strong white light(Hiwatashi and Ichikawa 1981, Tsubaki et al. 1987).It was therefore expected to inhibit PSP-toxin bio-synthesis; however, it stimulated PSP-toxin biosyn-thesis in vitro in both the dark and in the light. Acytochrome P450–dependent monooxygenase cantherefore be excluded as a candidate STX biosyn-thesis enzyme. On the other hand, flavin-dependentoxygenases are stimulated by carbon monoxide(Holmes and Rittenberg 1972, Murataliev and Feyer-eisen 1996) as well as by methylviologen (Holmesand Rittenberg 1972, Murataliev and Feyereisen1996). Methylviologen can replace flavin and ferre-doxin of oxidoreductases that utilize these naturalelectron carriers (Fujita and Peisach 1982, Hedde-rich et al. 1992, Michel et al. 1995, Shimada et al.1998, Schut et al. 2001). The natural electronsource for flavin-dependent dioxygenases arereduced nucleotides (NAD[P]H; Mason andCammack 1992), whereas 2OG dioxygenases require2OG (Prescott and Lloyd 2000). While NADH andNADPH moderately stimulated in vitro PSP-toxinbiosynthesis, a-ketoglutarate acted as a strong inhib-itor at concentrations greater than 0.5 mM(Table 2). Taken together, the stimulation of in vitroPSP-toxin biosynthesis by methylviologen, carbonmonoxide, and reduced nucleotides (Table 2) indi-cated that one or several oxygenases that containflavin, and possibly ferredoxin, as their prostheticgroups are involved in this pathway.

Cellular localization and regulation of PSP-toxin biosyn-thesis. Separation of the cytosolic fraction from themembrane fraction of the C. raciborskii T3 lysate byultracentrifugation revealed that all enzymesrequired for PSP-toxin biosynthesis were present inthe cytosolic fraction. No activity was detected in themembrane fraction, although the membrane frac-tion stimulated in vitro PSP-toxin biosynthesis of thecytosolic fraction and lysates, indicating that itcontained a specific limiting cofactor or metabolite.Although the enzymes were cytosolic, they appearedto sediment by centrifugation as biosynthetic activitywas the highest in the middle and bottom layers ofthe soluble fraction. This indicated that the biosyn-thesis enzymes may be large or form a multienzymecomplex.

Dinoflagellates and cyanobacteria that producePSP toxins are phototrophic organisms, and PSP-toxin production is regulated by light in bothgroups of organisms (Kim et al. 1993, Taroncher-Oldenburg et al. 1997, Yin et al. 1997). It is notknown whether this regulation occurs at thetranscriptional, translational, or posttranslational


level. The instant activation of in vitro PSP-toxinbiosynthesis by light suggested that it is regulated bya posttranslational mechanism. Activity of enzymes,such as phosphofructokinase, may be regulated bythe thiol:disulfide ratio (Gilbert 1982). Light-induced electron flow leads to an increase in thethiol:disulfide ratio, thus activating phosphofructok-inase. Other enzymes, such as sucrose-phosphatesynthase, are also activated in vitro by light-induceddephosphorylation (Huber and Huber 1992). Eithermechanism may be responsible for the light-inducedactivation of in vitro PSP-toxin biosynthesis.

Minimal biochemical requirements. Saxitoxin biosyn-thesis is complex and involves at least 10 differenttypes of biochemical reactions, each of which islikely to be catalyzed by a different enzyme or cata-lytic domain. Each enzyme family has specific bio-chemical requirements, including cofactors andmetal ions. Native enzyme extract was used forin vitro PSP-toxin biosynthesis, which contained anundefined and complex mixture of metabolites,including those required for the biosynthesis of PSPtoxins. One of the goals of the present study wastherefore to determine the minimum biochemicalrequirements for STX biosynthesis so that themolecular components of in vitro PSP-toxin biosyn-thesis reactions could be controlled and defined.

Desalting of enzyme extracts abolished PSP-toxinbiosynthesis activity. As determined by the concen-tration of remaining PSP toxins, desalting wasapproximately 93% efficient. Desalted enzymeextracts were supplemented with a wide range of co-factors and metal ions in numerous possible combi-nations to restore activity; however, this activity wasnot observed. Supplementation with the salt frac-tion, on the other hand, restored activity and indica-ted that desalting did not irreversibly inactivate theenzymes, but that an essential and unknown cofac-tor was removed from the lysate. Because the separ-ation range of Sephadex G-10 is 100 to 700 Da, theunknown cofactor must consist of a small molecule witha molecular weight of less than approximately 1000 Da.

The membrane fraction did not provide anyin vitro PSP-toxin biosynthesis activity but stimulatedthe activity in whole and cytosolic protein extractsand restored activity to desalted enzyme extracts,indicating that the insoluble fraction contained theunknown cofactor. Extraction of membrane frac-tions with chloroform, acetone, and methanolrevealed that the unknown cofactor was soluble inpolar solvents. In addition, it was not degraded byboiling for 15 min. These experiments indicatedthat the unknown cofactor was polar and heatstable. The fact that whole lysate or membrane frac-tions from non-PSP-toxin-producing C. raciborskiiAWT205, Anabaena sp. PCC7120, and E. coli did notstimulate in vitro PSP-toxin production by desaltedC. raciborskii T3 enzyme extract indicated that theunknown cofactor was not universal, but specific tothe PSP-toxin-producing strain.

The data presented in this study suggested thatcytochrome P450 and a-ketoglutarate-dependentoxygenases are not involved in PSP-toxin biosynthesis,but that oxygenation of STX precursor is catalyzedby one or several flavin-dependent oxygenases,which may involve ferredoxin as a natural electroncarrier. In addition, evidence was presented thatone or several PLP-dependent enzymes, such as arelative of aminolevulinate synthase and possiblyaminotransferase, take part in PSP-toxin biosynthe-sis. The fact that CARPB stimulated PSP-toxin bio-synthesis suggested that a carbamoyltransferase isalso involved. In vitro PSP-toxin biosynthesis wasactivated by light, indicating that it is regulated invivo by a posttranslational and light-dependentmechanism. The biosynthetic pathway of STX isunique and may involve novel enzymes that mayalso require novel cofactors. The requirement forthis specific cofactor has implications in the searchof the STX biosynthesis genes. In addition to thegene cluster encoding STX biosynthesis enzymes,organisms that produce STX analogs may possess agene cluster that is responsible for the biosynthesisof this unknown cofactor.

The present study validated in vitro PSP-toxin bio-synthesis as a tool to investigate its biochemicalrequirements and to make suggestions regardingthe identity of PSP-toxin biosynthesis enzymes.Future research will now be directed toward identi-fying the biosynthetic genes using a reverse geneticapproach that will target candidate gene families,including carbamoyltransferase, aminolevulinate syn-thase, and aminotransferase.

The authors wish to thank the ARC for their financial sup-port. Mass spectrometric analyses were performed by Dr. Rus-sell Pickford at the Bioanalytical Mass Spectrometry Facility(UNSW), supported in part by grants from the AustralianGovernment Systemic Infrastructure Initiative and MajorNational Research Facilities Program (UNSW node of theAustralian Proteome Analysis Facility) and by the UNSW cap-ital Grants Scheme and the Faculty of Engineering ResearchGrant for 2005.

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BIOCHEMICAL CHARACTERIZATION OF PARALYTIC SHELLFISH TOXIN BIOSYNTHESIS IN VITRO

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